The tick borne flaviviruses (TBFV) belong to the Family Flaviviridae, genus Flavivirus and comprise some of the most medically significant emerging and re-emerging viral pathogens. TBFV include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Powassan virus and Langat virus (LGTV). TBFV are transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. Other flaviviruses include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses. The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the arthropod and the mammal) critical to virus replication and pathogenesis. We study LGTV, a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. LGTV can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. Studies using LGTV will form the basis for work on the more virulent BSL-3 Powassan virus and BSL-4 TBEV. 1. Analysis of virus interactions with the invertebrate host. Ixodid ticks represent the natural reservoir of TBFV, are critical for virus persistence in nature, and are the major vector for infection of humans. Transmission of flaviviruses to humans occurs during tick feeding. We have developed a first generation microarray to investigate salivary gland transcriptional changes in Ixodes scapularis nymphs during feeding or after infection with LGTV. The immediate goal of this work is to identify tick salivary gland transcripts that play a role during feeding or for the replication or transmission of TBFV. The long-term goal of this work is to identify novel tick salivary gland genes that could be targeted for the development of vaccines that have the potential to disrupt tick feeding and/or flavivirus transmission. Comparison of fed and unfed ticks revealed a dramatic metabolic change reflected by up-regulation of 578 transcripts during feeding. A clear temporal pattern of gene expression changes was observed showing clusters of genes were specifically up-regulated at 1, 2 or 3 days of feeding. Comparison of LGTV-infected and uninfected ticks has identified 39 differentially regulated salivary gland transcripts over a time course of feeding. Differentially regulated transcripts have been annotated based on current knowledge from the Ixodes scapularis genome. These transcripts are classified as putative secreted proteins, lipocalins, Kunitz protease inhibitors, antimicrobial peptides, serpins, metalloproteases and transcripts of unknown function. In 2011, the results of these studies were submitted as a manuscript to Ticks and Tick Borne Diseases. in addition, we are currently generating a series of recombinant proteins based on transcripts of interest, and will investigate the effect of these proteins on tick borne flavivirus infection in vitro and in vivo. Since dendritic cells are thought to be initial targets of TBFV delivered by tick bites, our studies will focus on these cells. 2. Comparison of TBFV infection in vertebrate and invertebrate systems. TBFV are maintained in nature in an infectious cycle that involves both tick and vertebrate hosts. Thus, these viruses must successfully replicate in two very distinct systems. One basic difference between these two systems is that flavivirus infection of tick cells is persistent, whereas infection of mammalian cells is typically acute and cytopathic. The genetic determinants and attendant host factors underpinning these phenomena are not well understood. A. Viral determinants of pathogenesis in the arthropod vector and the mammalian host. In a previous study, we serially passaged Langat virus (LGTV) in either tick or mouse cell lines and found a limited number of amino acid mutations in these passaged viruses compared to the wildtype parental virus. Interestingly, the tick-passaged virus exhibited reduced neuroinvasiveness when injected IP into mice compared to the wildtype parental or mouse-passaged virus. Viral RNA from the brains of mice moribund following IP injection was characterized and five recurring amino acid changes were observed. Because of the location of these changes, we speculated that the amino acid mutations enabling neuroinvasiveness compensated for the mutations observed following passage in tick cells. In 2011, we have utilized a reverse genetics system to examine these amino acid mutations by introducing these specific amino acid mutations into the wildtype parental LGTV genome. The mutations of interest have been inserted into a full-length molecular clone of LGTV, and viruses have subsequently been rescued. The viruses are currently being studied for growth in tick and mammalian cells in vitro. An animal study protocol has been submitted to the IACUC for review and upon approval, we will begin examining the effects the introduced mutations have on the neuroinvasiveness of the LGTV. B. Microscopic comparison of TBFV infection in mammalian and tick cells. A key difference between TBFV infection of vertebrate and arthropod host systems is that infection of ticks is persistent and non-cytolytic, whereas infection of mammalian hosts is typically acute and cytopathic. We are investigating the nature of this difference to identify responsible host and viral factors. Flavivirus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. These rearranged membranes host virus replication and may protect replicative intermediates from intracellular antiviral systems. In the case of WNV and DEN, viral non-structural protein 4A (NS4A) has been implicated in the membrane rearrangements during infection. However, similar analysis has not been done with TBFV in tick cell lines. We are comparing virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal and electron microscopy. Electron microscopy has shown that TBFV-infected mammalian cells exhibit virus-induced vesicles, ER proliferation and accumulation of virions as early as 24 hrs post-infection. By 48 hrs post-infection, the virions assemble into paracrystalline arrays and large amounts of vesicles are observed. Significant cytopathic effect is observed at later timepoints. In the TBFV-infected tick cells, vesicle formation occurs later than in the mammalian cells and is not as prominent. In contrast to the mammalian cells, tubular profiles predominate instead of the sperical vesicles observed in the mammalian cells. No virions were detected in the tick cells at any time point post-infection. While this may simply be a consequence of the lower virus titer produced in the tick cells, these differences are currently being addressed using electron tomography to get a more detailed understanding of the virus-induced vesicle formation in the two different cell types. In addition to extending our work, in 2011, we have developed an in vitro model for persistent infection of tick cells and are examining these cells with the same array of techniques. Initial results, which are being quantitatively evaluated, suggest that the tubular profiles are more frequent in the persistently infected cells. The results from the microscopy experiments will form the groundwork for biochemical and genetic investigations.